: The two distinguishing features of B-1 cells (Ly-1+ B cells) compared to B-2 cells (Ly1- cells) is that the former are (hyper)responsive to phorbol ester and enter into S phase when incubated with PMA in the absence of other co-stimuli. B-1 cells also exhibit a defect in their ability to respond to ligands that-bind to surface immunoglobulin (sIg) in that they exhibit a normal Ca2+ mobilization response yet fail to hydrolyze phosphoinositides. Most importantly, B-1 cells do not enter into S phase in response to anti-Ig. The studies outlined in this application are divided into two major themes designed to further elucidate the molecular mechanisms underlying the aberrant responsiveness of B-1 cells to phorbol esters and ligands that bind to membrane immunoglobulin. The first aim constitutes two lines of study that are focused on elucidation of the molecular mechanisms underlying the responsiveness of B-1 cells to phorbol ester. First, experiments are proposed to identify differences in the expression and/or activity of PKC isoenzymes between B-1 and B-2 cells in order to determine whether selective expression or activity of a particular isoenzyme might lead to the responsiveness of B-1 cells to phorbol ester. PKC isoenzyme activity, protein expression and gene expression will be evaluated in B-1 and B-2 cells. In the event that specific differences are found in the activity and/or expression of specific isoenzymes, further studies will be carried out in which antisense oligonucleotides will be used to assess the dependence of the (hyper) responsive state of B-1 cells on the expression or activity of that particular isoenzyme. Based on the observation that B-1 cells exhibit an increased responsiveness to phorbol ester which is PKC dependent, a second series of experiments will be carried out to measure differences in downstream metabolic events that are regulated by PKC. Studies will be performed in which the composition of the B-1 cell AP-1 complex and its regulation will be compared to that of B-2 cells. The experiments proposed include a kinetic analysis of induction of the AP-1 complex, analysis of the composition of the AP-1 complex and competitive binding analysis to identify post-translational modifications of AP-1 complex components. The dependence of B-1 cell phorbol ester responsiveness on increased induction of AP-1 or on the differential composition of the complex will be assessed using antisense oligonucleotides. The second major component of the project involves studies to elucidate the molecular mechanisms underlying defective sIg- mediated signaling in B-1 cells.These experiments are primarily focused on delineation of the differences in tyrosine phosphorylation in B-1 cells as compared to B-2 cells. Differences in tyrosine phosphorylation in B-1 cells in response to anti-Ig will be analyzed. Specific attention will be paid to the question of PLC phosphorylation in these cells in view of the inability of B-1 cells to hydrolyze phosphoinositides following sIg ligation. Additional experiments will determine whether there are differences in the expression of specific src family protein tyrosine kinases in B-1 cells that might explain their inability to respond to anti-Ig. Activation of src kinases following anti-Ig treatment will be monitored as well. In view of the fact that aberrant tyrosine phosphorylation is observed in B-1 cells following anti-Ig treatment, it is equally possible that alterations in protein tyrosine phosphatase activity might be involved. In view of this, the expression and activity of CD45 isoforms on B-1 cells will be examined.Analysis of CD45 phenotype will be carried out using mAbs directed against specific N-terminal exons of the molecule that are alternatively spliced to form the various isoforms. To further characterize CD45 isoform expression, RT-PCR will be carried out to analyze mRNA levels for specific CD45 isoforms. The possibility exists that CD45 expression in B-1 cells is not appreciably different from that in B-2 cells but that there is a difference in post-translational modification of CD45 in B-1 cells that affects its catalytic activity. Thus, experiments will be carried out to determine whether there are differences in the activity of CD45 in B-1 cells. The final issue that will be examined is the composition of the sIg receptor complex. Experiments designed to assess the association of MB-1 and B29 with the antigen receptor and their phosphorylation will be conducted.